高尔基体蛋白GOLPH3通过mTORC1信号通路调控溶酶体发生的分子机制

Golgi protein GOLPH3 regulates lysosomal biogenesis through mTORC1 signaling

目的探究哺乳动物细胞中高尔基体磷酸化蛋白3(GOLPH3)通过mTORC1信号通路调控溶酶体发生的分子机制。方法利用CRISPR Cas9建立GOLPH3敲除稳定细胞株。通过Western blot分析比较对照细胞与GOLPH3敲除细胞的mTORC1活性、TFEB(转录因子EB)以及pTEFB水平,进一步用免疫荧光方法标记溶酶体联系膜蛋白LAMP1,比较两种细胞中溶酶体数量的差别。结果细胞中敲除GOLPH3将显著抑制mTORC1活性,减少细胞质中pTEFB水平,增加活化TFEB的细胞核定位,促进溶酶体的生物发生。结论高尔基体蛋白GOLPH3通过mTORC1信号通路调控溶酶体发生。

Objective To explore the mechanism of Golgi phosphoprotein 3(GOLPH3)regulating lysosomal biogenesisviamTORC1 signaling.Methods GOLPH3 knock-out(GOLPH3 KO)stable cell line was constructed by CRISPR Cas9.mTORC1 activity and the levels of TFEB and p-TFEB in the control cells and GOLPH3 KO cells were compared through Western blot.Further,lysosome-associated membrane protein LAMP1 was labelled by the means of immunofluorescence and the number of lysosomes in the control cells and GOLPH3 KO cells was compared.Results GOLPH3 KO suppressed mTORC1 activity significantly,decreased the cytoplasm level of pTFEB,increased the nuclear localization of active TFEB,and promoted the lysosome biogenesis.Conclusion Golgi protein GOLPH3 regulates lysosome biogenesis through mTORC1 signaling.